Cardiac electrical and mechanical functions depend upon homeostatic ion gradients that are regulated by a number of transmembrane ion transport mechanisms including L-type Ca2+ channel current (ICa.L) and Na+/Ca2+ exchange current (INa/Ca). Alterations in these currents, such as those induced by neurohumoral agents (e.g. cytokines), can change cardiac function in both the normal and diseased heart. Proinflammatory cytokines such as interleukin (IL)-1beta and tumor necrosis factor (TNF)- alpha have been associated with injury- and immune-mediated decreases in cardiac function; however, little is known about the cellular mechanism(s) underlying this cytokine-induced cardiac depressant effect. Therefore, the long-term goal of this research is to define the signal transduction cascades of endogenous neurohumoral peptides (including IL-1beta and TNF- alpha) that modulate myocardial electrical activity and contraction under physiological and pathological conditions. Ceramide, which is produced by IL-1beta and TNF-alpha stimulation, has been shown to mediate many biological effects of these two cytokines in non-cardiac cells, and our preliminary studies show that ceramide is involved in the IL-1beta-induced suppression of ICa.L in adult rat ventricular myocytes. However, the mechanism by which ceramide suppresses ICa.L and other transporters that modulate cardiac contractile function remains undefined. Thus, the goal of this proposal is to elucidate the role of ceramide in cardiac cell function. The following specific aims will be addressed. Specific Aim 1 will test the hypothesis that ceramide suppresses ICa.L, INa/Ca and the Ca2+ transient, thereby suppressing cardiac contractility. Specific Aim 2 will test the hypothesis that phosphorylation of specific proteins is required for the effects of ceramide on these membranes currents and contraction. Specific Aim 3 will test the hypothesis that mitogen-protein (MAP) kinase and protein kinase C (PKC-zeta) are involved in the cardiac effects of ceramide. Using adult rat ventricular myocytes as a model, this project will utilize multiple technical approaches including patch-clamp techniques, biochemical and molecular assays (e.g. Western blot analysis), contraction measurement (e.g. edge-detection techniques), and fluorescent microscopy to study the mechanism by which ceramide mediates changes in cardiac cell function. Results of this study will add significant new information concerning the pathophysiological role of ceramide in the modulation of cardiac function. Thus, the results will also offer insights into the possible development of specific pharmacological strategies to treat the cardiac dysfunction associated with immunologically-mediated disorders and cancer treatment by cytokines.